Purine derivatives having pharmacological action

ABSTRACT

Compounds of formula I ##STR1## wherein R 1  is OH or NH 2  and R 2  is H or NH 2 , R is H or lower alkyl, A is an alkyl chain optionally interrupted by heteroatoms and B is an amino acid, a di- or tri-peptide, are useful in human therapy.

The present invention relates to purine derivatives having animmunomodulating, antiviral activity and on Central Nervous System, to aprocess for the preparation thereof and to pharmaceutical compositionscontaining them.

The derivatives according to the invention can be presented by thefollowing tautomeric general formula I ##STR2## wherein R₁ is OH or NH₂and R₂ is hydrogen or NH₂, with the proviso that R₁ and R₂ cannot beboth NH₂ groups, R is hydrogen or C₁ -C₆ alkyl, A is a group of formulaII

    --(CH.sub.2).sub.m --X--(CH.sub.2).sub.n --                (II)

or a group of formula ##STR3## wherein X is oxygen, sulphur or seleniumm is an integer from 0 to 4;

n is an integer from 2 to 4;

p is an integer from 0 to 8;

R₃ is hydrogen or C₁ -C₆ alkyl;

B is a residue of a natural amino acid of the l, d or dl series or a di-or tri-peptide residue in which the amino acids which compose it areselected from the group consisting of alanine, arginine, glycine,isoglutamine, leucine, lysine, methionine, ornithine, proline andserine, with the proviso that, when A is a residue of formula III, B isalways a di- or tri-peptide residue as above defined.

In case amino acid B or N-terminal amino acid of the di- or tri-peptideis a basic amino acid (arginine, ornithine, lysine), the carbamate bondcan involve either the amino group which is at the l-position to thecarboxy group or the amino group at the w-position to the carboxy group.

The present invention also relates to non toxic salts of compounds offormula I.

European patent N. 0 077 660 discloses purine derivatives in which anamino acid is linked to the purine ring at the 9-position by means ofcarbonyloxyalkyl or carbonylethylcarbonyloxyalkyl bridges.

The derivatives according to the present invention show, in comparisonto the known compounds, a different and surprising pharmacologicalactivity range, particularly immunostimulating activity, antiviralactivity and activity on Central Nervous System, so that they arevaluable for use in human therapy.

Preferred compounds according to the invention are:

(a) compounds in which R is hydrogen, A is a group of formula II whereinm is 0 or 1, X is oxygen, n is 2 and B is an amino acid residue selectedfrom the group consisting of arginine, methionine, leucine or theresidue of a di- or tri-peptide selected from the group consisting ofleucyl-methionine, ornithyl-ornithine, glycyl-leucyl-methionine,leucyl-seryl-arginine, lysyl-arginine, alanyl-isoglutamine,lysyl-lysine, histidyl-leucyl-methionine, propyl-arginine;

(b) compounds in which R is hydrogen, A is a residue of formula III inwhich p is 2 or 3, R₃ is hydrogen and B is a di- or tri-peptide selectedfrom the above defined group.

Compounds I are prepared by reaction of a compound of formula IV:##STR4## wherein R₁, R₂, R and A are as above defined, with phosgene andsubsequent condensation with an amino acid or dior tri-peptides offormula H₂ NB according to per se known procedures.

Compounds of formula IV in which R₁ is OH, R₂ is hydrogen or NH₂ and Ais a residue of formula III wherein R and R₃ are hydrogen, are preparedaccording to the procedures described in EP-B-77460. Compounds IVwherein R₁ is OH, R₂ is hydrogen and A is a residue of formula III canbe prepared according to the process described in Italian Pat.Application N. 19083 A/87.

Compounds IV in which R₁ is OH and R₂ is NH₂ can be obtained accordingto the following reaction scheme: ##STR5## wherein Q is a residue offormula ##STR6## in which R and A are as above defined.

Compounds IV in which R₂ is hydrogen and R₁ is NH₂, on the contrary, areobtained from compounds of formula V ##STR7## wherein R and A are asabove defined, by reaction with liquid NH₃ at high temperature.

For the preparation of compounds IV in which A is a residue of formulaII, reaction schemes analogous to the ones reported above and the onesknown for the preparation of purine systems can be conveniently used.Thus, for example, 4,6-dichloro-5-amino-pyrimidine can be reacted with acompound for formula H₂ N(CH₂)_(m) --X--(CH₂)_(n) OH wherein X, m and nare as above defined, to obtain the intermediates of formula VI:##STR8## which can then be transformed into corresponding compounds I bymeans of an appropriate combination of known reactions, analogous to theabove reported ones and anyhow known to those skilled in the art.

The following examples further illustrate the invention without limitingits spirit and scope.

EXAMPLE 1N-[N-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-leucyl]-L-methionine##STR9##

0.850 g (0.0032 mole) of leucyl-methionine and 0.824 g (0.0096 mole) ofsodium bicarbonate were dissolved in 40 ml of bidistilled water, in a150 ml flask. To the resulting aqueous solution, having pH=8, asuspension of 1.05 g (0.0032 mole) of9-[5-chlorocarbonyloxy)pentyl]hypoxanthine hydrochloride in 40 ml oftoluene was added in ice bath.

The reaction mixture was left to react at room temperature undermagnetic stirring for 5 hours. After that the two phases were separated;the solvent was evaporated from the aqueous phase to obtain 1.8 g ofcrude compound which was chromatographed on a silica gel column using asthe eluent first a 1:1 ethyl acetate/ethanol mixture, then methanol.1.450 g of compound were thus obtained.

    ______________________________________                                                    theorical                                                                            found                                                      ______________________________________                                        C             51.75    51.82                                                  H              6.71     6.70                                                  N             16.45    16.38                                                  ______________________________________                                    

EXAMPLE 2 N² -[N⁵-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-ornithil]-L-ornithine and N⁵-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-N² -L-ornithil-L-ornithine##STR10## To a suspension of 1,05 g (0,0032 mole) of9-(5-chloro-carbonyl-oxy-pentyl)-hypoxanthine hydrochloride in 40 ml oftoluene, a solution of 1.68 g (0,0032 mole) di ornithyl-ornithinemonohydrochloride-trihydrobromide and 1,9 g (0,0224 mole) of sodiumbicarbonate in bidistilled water at pH=8 was added, under magneticstirring in ice bath. When the addition was over the reaction mixturewas left to stand for 20 hours at room temperature. Controls werecarried out by TLC (Thin Layer Chromathography), using methanol as theeluent. At the end of the reaction the two phases were separated, theaqueous phase was evaporated under vacuum and the resulting crudecompound was chromatographed on silica gel, using methanol as theeluent. The mixture of the two products was thereby obtained, whichproducts were separated by means of preparative HPLC.

    ______________________________________                                        theorical         found   found                                               ______________________________________                                        C      51,03          51,06   50,98                                           H       6,87           6,89    6,81                                           N      22,66          22,65   22,63                                           ______________________________________                                    

EXAMPLES 3-9

The compounds hereinbelow reported were obtained by means of ananalogous process, using corresponding amounts of the appropriatepeptide.

EXAMPLE 3N-[N-[N-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]glycyl]-L-leucyl-methionine

    __________________________________________________________________________     ##STR11##                                                                                        theorical                                                                          found                                                __________________________________________________________________________             C          50,81                                                                              50,77                                                         H           6,52                                                                               6,59                                                         N          17,27                                                                              17,22                                                __________________________________________________________________________

EXAMPLE 4 N²-[N[N-[5-(hypoxanthin-9-yl)penthyloxycarbonyl]-L-leucyl]-L-seryl-arginine

    __________________________________________________________________________     ##STR12##                                                                                       theorical                                                                          found                                                 __________________________________________________________________________               C       50,15                                                                              50,12                                                            H        6,79                                                                               6,73                                                            N       22,48                                                                              22,44                                                 __________________________________________________________________________

EXAMPLE 5 N² -[N⁶-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-lysyl]-L-arginine and N² -[N²-[5-hypoxanthin-9-yl)pentyloxycarbonyl]-L-lysyl]-L-arginine

    ______________________________________                                         ##STR13##                                                                     ##STR14##                                                                            theorical      found   found                                          ______________________________________                                        C       50,17          50,19   50,16                                          H        6,95           6,89    6,93                                          N       25,42          25,39   25,44                                          ______________________________________                                    

EXAMPLE 6 N²-[N-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-alanyl]-L-isoglutamine

    __________________________________________________________________________     ##STR15##                                                                                   theorical                                                                          found                                                     __________________________________________________________________________            C      49,03                                                                              49,00                                                             H       5,84                                                                               5,87                                                             N      21,05                                                                              21,06                                                     __________________________________________________________________________

EXAMPLE 7 N² -[N⁶-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-lysyl]-L-lysine and N⁶-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-N² -L-lysyl-L-lysine

    __________________________________________________________________________     ##STR16##                                                                     ##STR17##                                                                            theorical      found                                                                             found                                              __________________________________________________________________________    C       52,86          52,89                                                                             52,84                                              H        7,33           7,31                                                                              7,29                                              N       21,43          21,40                                                                             21,45                                              __________________________________________________________________________

EXAMPLE 8 N-[N-[N²-5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-histidyl]-L-leucyl-L-methionine

    __________________________________________________________________________     ##STR18##                                                                                     theorical                                                                          found                                                   __________________________________________________________________________              C      51,92                                                                              51,88                                                             H       6,38                                                                               6,35                                                             N      19,45                                                                              19,47                                                   __________________________________________________________________________

EXAMPLE 9 [N-[(hypoxanthin-9-yl)pentyloxycarbonyl]prolyl]-arginine

    __________________________________________________________________________     ##STR19##                                                                                  theorical                                                                          found                                                      __________________________________________________________________________            C     50,86                                                                              50,83                                                              H      6,40                                                                               6,41                                                              N     24,25                                                                              24,29                                                      __________________________________________________________________________

EXAMPLE 10

Preparation of N²-[2-[2-(hypoxanthin-9yl)ethoxy]-ethoxy-carbonyl]arginine

a) Preparation of4-chloro-5-amino-6-[(2-(2-hydroxyethoxy)-ethyl]amino-pyrimidine.

508 ml di n-pentanol, 25 g (0,152 mole) of4,6-dichloro-5-aminopyrimidine, 36,25 ml (0.152 mole) of tributylamine,11 ml (0,152 mole) of 2-(2-aminoethyoxy)ethanol and KI in catalyticamounts were placed into a three neck flask provided with refluxcondenser and mechanical stirrer, for 24 hours. The progress of thereaction was checked by TCL, using ethyl acetate as the eluent. At theend of the reaction pentanol was evaporated under reduced pressure andthe resulting crude reside was chromatographed on silica gel using 9:1chloromethane/ethanol as the eluent.

The compound was crystallized from ethyl acetate. M.P.=121° C.

b) Preparation of 9-[2-(2-hydroxyethoxy)ethyl]hypopxanthine.

10 g (0,043 mole) of compound a) in 150 ml of formic acid were placedinto a 250 ml flask. The reaction was refluxed for 24 hours. Theprogress of the reaction was controlled by TLC using 8:2ethanol/triethylamine as the eluent.

AT the end of the reaction, formic acid was evaporated under reducedpressure and the residue was crystallized from ethanol.

Reaction yield: 55%.

M.P.>200° C.

c) Preparation of 9-[2-[2-chlorocarbonyloxy)ethoxy]ethyl]-hypoxanthinehydrochloride.

3 g (0,0133 mole) of compound b) were reacted with 30 ml of 20% phosgenein toluene in 80 ml of anhydrous toluene, under magnetic stirring. It isadvisable to drop the phosgene solution. The reaction was controlled byTLC using ethyl acetate/ethanol in 1:1 ratio as the eluent. In order tovisualize the product in the plate, a reaction sample was treated with atert-butylamine excess to obtain corresponding urethane. At the end ofthe reaction solvent was evaporated off under reduced pressure to obtainthe crude product which was directly reacted for:

d) preparation of N²-[2-[2-(hypoxanthin-9-yl)-ethoxy-ethoxy-carbonyl]arginine.

0,927 g (0,0053 mole) of L-arginine was dissolved in 10 ml of water,under magnetic stirring. 0,86 g (0,002 mole) of compound C, suspended in10 ml of toluene, was added to the above solution. The reaction mixturewas left under magnetic stirring for 5 hours.

At the end of the reaction the two phases were separated and the aqueousphase was evaporated under reduced pressure. The resulting crude residuewas purified on a silica gel column, using methanol as the eluent. 0,5 gof compound was obtained.

EXAMPLE 11

Using 2-(2-aminoethyl)thio)ethanol, by means of a process analogous tothe above one, the following compound was prepared:

N² -[2-[2-(hypoxanthin-9-yl)-ethylthio]ethoxycarbonyl]-L-arginine,according to the following reaction scheme: ##STR20##

Biological activity of the above exemplified compounds was studied, andthe compounds according to the invention proved to be active asimmunomodulators, neurotransmitters and antiviral agents. Particularly,the compounds of the invention turned out to be active in the followingtests, showing an interaction on the immunosystem:

a) in vitro generation of Natural Killer cells from bone marrowprecursors;

b) in vivo boosting of Natural Killer cells activity;

c) in vitro cytotoxicity against YAC-l target cells:

d) transplant of bone marrow in mice;

e) macrophages activation.

Some of the compounds of the invention (example 3 and analogous), showedneurokinin-like activity in several in vitro models. They possiblely acton neurokinin receptors such as NK-P, NK-A and NK-B.

Moreover, the compounds of the invention proved to be active in in vitroantiviral activity tests on Vescicula Stomatitis Virus (VSV) and onencephalomyocarditis in L929 cells.

From what has been above reported, it is evident that the compounds ofthe invention can be advantageously used in human therapy of diseaseshaving viral, tumor and bacterial origin, of neurologic syndromes ordifferent pathologies in which immunosystem is recognizedly involved.

The present invention also relates to all the industrially applicableaspects related to the use of compounds I as therapeutical gent. Thus,an essential object of the invention is provided by pharmaceuticalcompositions containing as the active ingredient compounds I alone or inadmixture with a pharamaceutical carrier, in form of tablets,sugar-coated pills, capsules, powders, granules for reconsititution inoral solutions or suspensions, syrups, injection vials, etc.

The active ingredients can be alone in capsules. Otherwise, they can beformulated using traditional pharmaceutical carriers, for exampleexcipients such as lactose or talcum, granulation agents such asmethylcellulose and/or surface active agents such as polyoxyethylenestearate; preservatives such as ethyl p-hydroxybenzoate and possibleyflavoring agents.

The pharmaceutical compositions of the present invention can preferablybe formulated in form of unitary dosage containing 1 to 1000 mg of acompound of formula I in admixture with a pharmaceutical carrier. Saidunitary doses can be administered one to more times a day, depending onthe pathology and the conditions of the patient.

We claim:
 1. A compound selected from the group consistingof:N-[N-5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-leucyl]-L-methionine;N² -[N⁵-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-ornithyl]-L-ornithine; N⁵-[5-hypoxanthin-9-yl)pentyloxycarbonyl]-N² -Lornithyl-L-ornithine;N-[N-[N-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]glycyl]-L-leucyl-methionine;N²-[N-[N-[5-(hypoxanthin-9-yl)penthyloxycarbonyl]-L-leucyl]-L-seryl-arginineN² -[N⁶ -[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-lysyl]-L-arginine; N²-[N² -[5-(hypoxanthin-9-yl)penthyloxycarbonyl]-L-lysyl]-L-arginine; N²-[N-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-alanyl]-L-isoglutamine; N²-[N⁶ -[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-lysyl]-L-lysine; N⁶-[5-(hypoxanthin-9-yl)penthloxycarbonyl]-N² -L-lysyl-L-lysine; N-[N-[N²-[5-(hypoxyanthin-9-yl)penthyloxycarbonyl]-L-histidyl]-L-leucyl-L-methionine;N² -[N-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-proly]-L-arginine; 2.The method of treating a subject affected by leukemia or a solid tumoror a subject in need of bone marrow transplant which consists ofadministering to said subject the compoundN-/N-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-leucyl]-L-5 methionine.